Abstract: The specific goal of our proposal is to understand the mechanism of Runt-related factor-2 (Runx2)-mediated autophagy in bone metastasis of breast cancer. Bone is a common target site of distant metastasis of breast cancer. Metastasis to bone results in severe bone loss, fractures, and death of more than 30,000 individuals each year. Recent studies show that metastatic cancer cells induce autophagy to survive stress conditions. Autophagy is a highly regulated mechanism by where cytoplasmic components are degraded by the lysosome for cell survival during starvation. Despite the recent progress, the molecular mechanisms of autophagy during bone metastasis remain poorly understood. Accordingly, our long-term goal is to define the regulation of autophagy and understand how this regulatory mechanism can be utilized to treat bone metastasis. Utilizing a bone metastatic breast cancer mouse model, we discovered that autophagy is enhanced by Runx2. We demonstrated that Runx2 facilitates acetylation of microtubules (MTs), a critical step in trafficking of autophagosomes. Previously, we reported that Runx2 promotes metastasis of breast cancer cells. Based on these findings, our central hypothesis is that metastatic breast cancer cells survive in the bone microenvironment via Runx2-dependent autophagy. To test this hypothesis we propose the following Aims: 1) Define the mechanism of Runx2-mediated microtubule acetylation during autophagy. We will examine the impact of Runx2-mediated acetylation of ?-tubulin subunits of MTs on autophagy. We will determine the interaction among Runx2 and co-factors regulating acetylation of MTs. For this, we will use bone metastatic breast cancer cells expressing altered levels of Runx2 or HDAC6. 2) Determine the impact of Runx2-mediated autophagy on bone metastasis. We will examine the Runx2-mediated autophagy function by altering Runx2 levels in combination with (i) inhibition of HDAC6 activity, (ii) expression of an acetylation-deficient mutant of ?- tubulin subunit of MTs, and (iii) treatment with a pharmacologic inhibitor of autophagy in xenograft and syngeneic tumor mouse models of bone metastasis. Our proposed studies will reveal novel survival mechanisms of bone metastatic breast cancer cells and will identify critically needed targets for treatment of metastatic bone disease.